A method of non destructive monitoring of biological processes in microfluidic tissue culture systems

ABSTRACT

Microfluidic “organ-on-a-chip” devices have been developed with the aim to replicate human tissues in vitro. However, there is no option to quantitatively monitor biological processes that take place within the chip, over time. Destructive methods in order to analyze, tissue formation, gene expression, protein secretion etc. require the harvest of the “tissue” at a certain time point. Described herein are methods and compositions for non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest.

FIELD OF THE INVENTION

Described herein are methods and compositions related to imaging of stem cells and cells undergoing differentiation without sample manipulation.

BACKGROUND

Bones consisting of mineralized bone tissue also consists of bone marrow, nerves and blood vessels. Development and homeostasis of bone relies heavily on communication between cells in the tissues as regulated by the bone environment. Bone is an active tissue maintained by bone cells such as osteoblasts that form bone and osteoclasts that resorb bone, and it is now understood that mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more. Additionally, within the collagen and mineral matrix osteocytes are also embedded and respond to the bone environment. The balance between these cells is necessary to maintain bone function. Studying bone is a challenging field due to microarchitecture defining the bone environment, which involve the intricately dense structural composition of the bone morphology. Unlike other tissues that can be processed and prepared for experiments, including cultured cell lines, working with bone is difficult. Studying intracellular dynamics of the bone cells embedded within the mineralized tissue has proven to be a challenging task.

Compounding these challenges related to underlying properties of the cellular material, imaging cells at subcellular level within the bone environment is very difficult. Paraffin tissue slices are a long standing conventional approach to evaluate microarchitecture and bone morphology. However, sample manipulation leads to changes in biochemical properties of antigenicity and mineral structure. Newer strategies to image cells within the bone such as MM, Micro-CT or Ultrasound can image bone structure and recently cells, however these techniques are limited by their low resolution at the cellular level given the surrounding physiological environment.

Recent “organ-on-a-chip” technologies represent new and exciting opportunities for bone research. These devices include a microfluidic cell culture apparatus that is a more physiologically relevant in vitro model than cells cultured in dishes. Importantly, providing for continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physiology allow for the culturing of bone cells in a format mirroring their physiological environment. By studying bone cell function and response in this manner, a 3D environment can reveal completely different cellular dynamics compared to 2D cultures. The availability of cellular tissue material in this format further provides new avenues for apply imaging approaches of bone microarchitecture to identify features previously unavailable in tissue cultures or at insufficient resolution in vivo. Real time imaging of the bone marrow niche within bone and fluorescent imaging of cells within the bone marrow niche has reportedly been achieved. Recent advancements in imaging techniques allows for the identification of osteocytes embedded in the bone matrix. However, determining the localization of cell types and protein expression dynamics of single cells within the bone is still very difficult. And more research is needed to identify intracellular protein activities of the cell bodies embedded within mineralized matrix.

Described herein is the use of non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest the tissue. Such methods can provide valuable data on developing tissues and their response to pharmaceutical, chemical and environmental agents.

SUMMARY OF THE INVENTION

Described herein is a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, the one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled 4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, further culturing of MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.

Also described herein is a method of detecting secreted extracellular macromolecules in a microfluidic device including providing a microfluidic device including stem cells, applying one or more pulse sequences to the microfluidic device; and, detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are pluripotent stem cells (pSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further detection of pulse sequence signal intensity.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Organ-on-chip dimensions and setting of the flow system. The flow was set to 30 μl/h and the media in the reservoirs was replaced or refilled twice a week.

FIG. 2. Cell survival and proliferation on the organ-on-chip. FIG. 2A. Micrographs of the cells on the chip grown for 3 weeks in osteogenic conditions. FIG. 2B. bioluminescent imaging (BLI) images taken on Day 0. FIG. 2C. Quantitative analysis BLI that was done twice a week for 3 weeks. Bars indicate standard deviation, n=5, *p<0.05; ***P<0.001.

FIG. 3. Osteogenic differentiation at Week 3 measured with two probes (OsteoSense and BoneTag) using two different imaging systems: fluorescent imaging system (IVIS, Perkin Elmer) and Near Infrared imaging system (Odyssey® CLx, Li-Cor). FIG. 3A. FLI images of chips incubated with BoneTag and in FIG. 3B the labeling was quantified FIG. 3C. using fluorescent imaging IVIS Live staining where live cells arte stained with FITC (green) dye and OsteoSense is depicted in red and imaged using confocal microscopy, 10× magnification. In FIG. 3D, labeling was quantified, bars indicate standard deviation, n=5, *p<0.05; ***P<0.001.

FIG. 4. Immunostaining of osteogenic differentiation of MSC-BMP2 in Organ-on-chip. The chips were sectioned using vibratome across the channels. The sections were stained using with immunofluorescent staining against the osteogenic markers osteocalcin and bone sialoprotein (BSP), and imaged using confocal microscopy.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

In recent years, microfluidic devices have been developed with the aim to replicate human tissues in vitro. These systems, also called microfluidic chips or “organ-on-a-chip”, have the potential to serve as an alternative for animal models that are used to test pharmaceutical, chemical and environmental agents. The microfluidic chips are attractive for biomedical research and drug discovery due to low cost and ethical considerations compared to animal models. A variety of examples are described in Bhatia and Ingber, “Microfluidic organs-on-chips.” Nat Biotechnol. 2014 August; 32(8):760-72, which is fully incorporated by reference herein.

An important caveat of the “‘chips” is that currently there is no option to quantitatively monitor biological processes that take place within the chip, over time. To date, researchers are using destructive methods in order to analyze tissue formation, gene expression, protein secretion etc. These methods include histology, immunofluorescence or PCR and require the harvest of the “tissue” at a certain time point. The use of non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest the tissue would be a significant improvement in the field. Such methods can provide valuable data on developing tissues and their response to pharmaceutical, chemical and environmental agents.

Mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts, chondrocytes, fibroblasts, adipocytes, tenocytes, nucleus pulposus cells and more. In situ imaging, both in non-living and living specimens, have provided new insights, but for the above described reasons, quantitative experimental data requires destructive processing that may introduce bias, and lack temporal and spatial resolution. In this regard, microfluidic organ-on-a-chip coupled with non-destructive labeling and imaging techniques may allow precise capture of MSC, osteoblast and osteocyte cell populations in micro and ultrastructure in 2D and 3D. Live cell imaging techniques which are able to track structural morphology and cellular differentiation in both space and time combined with the latest biochemical assays and microfluidic imaging techniques can provide further insight on the biological function of MSC, MSCs, osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells.

Existing techniques for imaging of cells in skeletal and other tissues has proved challenging due to the need to develop methodologies for sectioning specimens, labeling or imaging of specimens or to develop protocols for decalcifying specimens to enable conventional sectioning and imaging techniques to be used. Current imaging approaches rely mainly on histological stains combined with conventional light microscopy. Confocal imaging approaches allows for three-dimensional (3D) imaging in situ within the bone environment. In contrast to inherently two-dimensional (2D) imaging techniques such as light microscopy, confocal microscopy stacks optical sections at different focal planes to generate a three-dimensional (3D) representation of the sample. Endogenous (auto)fluorescence of the bone tissue can be used to provide contrast for confocal microscopy measurements. More often, various fluorescent staining agents are used in conjunction with modern confocal laser scanning microscopy (CLSM), such as rhodamine and fluorescein, which can be incubated with undecalcified bone sections. More specific staining agents, such as fluorescein isothiocyanate (FITC)-conjugated phalloidin and DAPI, label the actin skeleton and/or DNA of cell nuclei in such a way that the components cells can be directly imaged and separately displayed in 3D

However, a major drawback with CLSM is the limited maximum focal plane depth of around 100-150 μm. Additionally, CLSM is tainted with image artifacts, such as signal attenuation with increasing focal plane depth or aberrations due to refractive index mismatch. Such artifacts are absent in (conventional) X-ray absorption-based computed tomography (CT). Micro-computed CT (μCT) and 3D morphometric measures to quantify trabecular microarchitecture have laid the foundations for μCT to become a standard for bone morphometry. In bone research, the standard application of desktop μCT systems with typical voxel sizes in the order of 5-100 μm is a core approach for quantitative characterization of whole bone geometry. Synchrotron radiation-based CT allows for imaging of bone microstructure, canal networks, as well as study of populations such as osteocytes within bone. Most recently, optimized imaging protocol for SR CT provides spatial resolution closer to the diffraction limit of visible light at a few hundred nanometers. The recent availability of desktop μCT scanners with voxel sizes below 1 μm allow for new opportunities for imaging.

Over the past two decades or so, technologies for imaging of living cells using light and confocal microscopy have advanced at a rapid rate. Coupled with enhanced green fluorescent proteins (GFPs) and a seemingly limitless array of fluorescent imaging probes has made it possible to image almost any intracellular or extracellular structure or protein in living cells and tissues. A large selection of fluorescent probes and reagents are commercially available to the researcher for investigating biological events in living cells, including fluorescent antibodies, kits for fluorescently labeling proteins of interest, dyes for cell and nuclear tracking, probes for labeling of membranes and organelles, fluorescence reagents for determining cell viability, probes for assessing pH and ion flux and probes for monitoring enzyme activity, etc. In addition, a variety of GFP-derived fluorescent protein vectors are available that can either be used as reporter constructs or to generate fusion constructs with a protein of interest. These enable the live monitoring of gene expression and protein localization in vivo, and in real time.

The traditional approach of collecting “static” images of fixed or post mortem cells and tissues provides a snapshot view of events at a single fixed point in time. However, this inherently overlooks the dynamic aspects of the biology being examined. In contrast, live cell imaging enables the visualization of temporal changes in living specimens and can reveal novel aspects of the biology that may not otherwise have been appreciated. Additionally, the datasets generated from time-lapse imaging are information rich and can be interrogated quantitatively to enable measurement of cellular, subcellular and tissue dynamic events as a function of time

Although these approaches are leading to exciting discoveries that are advancing our understanding of biological systems, there are several limitations that need to be acknowledged. Firstly, fluorescent probes may perturb or alter the biology being examined. Validation studies are needed to make sure that the fusion protein still functions similarly to the wild type form. It is also advantageous to confirm findings with more than one type of imaging probe if possible. For example, a GFP fusion protein can be used for in vivo localization of a specific protein and key data can be confirmed using a fluorescence-conjugated antibody against the same protein. When developing live cell imaging protocols, there is always a compromise between obtaining a high enough signal-to-noise ratio to enable quantitative measurements and to obtain sufficient image resolution, while at the same time avoiding phototoxic effects to the cells. Therefore, to ensure cell viability, the researcher may have to accept a lower image quality and resolution than would be acceptable for equivalent images of fixed specimens. Nevertheless, technologies such as multiphoton fluorescence microscopy can increase the depth of tissue penetration for live cell imaging applications and reduce phototoxicity by using a longer wavelength light to excite fluorophores. These instruments are becoming more widely used for live imaging applications due to their advantages over conventional widefield and confocal microscopy systems.

Recently, live cell imaging approaches have been applied to the study of MSCs, osteoblasts and osteocytes. Organ cultures of neonatal calvaria from mice have provided a useful model for imaging the dynamic properties of osteocytes. Another way in which this model can be used for imaging osteocyte dynamics is by using long term cultures of MSCs and osteoblasts. These cells differentiate when cultured under mineralizing conditions to form mineralized nodules in which the transition to the osteocyte-like phenotype can be monitored by fluorescent labeling or radiolabeling. To gain maximum information, imaging of these can be combined with other fluorescent probes, such as alizarin red to monitor mineral deposition. Live cell imaging studies as applied to investigating osteocyte biology are still in their infancy. In addition to revealing the dynamic properties of MSCs, osteoblasts and osteocytes, identifying the underlying intracellular signaling pathways, such as calcium oscillations, monitoring the temporal integration of osteocyte differentiation and mineralization, live imaging studies have considerable potential to address many as yet unresolved questions in osteocyte biology.

Most importantly, biochemical data characterizing the precise role of MSCs, osteoblasts and osteocytes in bone remodeling remains severely limited. A number of in vivo models have been developed to study their function. Existing technologies typically harvest large osteocyte populations and employ technologies which provide a comprehensive assessment of a large number of genes which are both up-regulated and down-regulated in response mechanical stimulation. For example, to comprehensively assess osteocyte gene expression in a mouse model for load induced bone adaptation, current state-of-the-art approaches extract large populations of osteocytes from loaded bone and perform micro-array-analysis to quantify the expression levels of tens of thousands of different genes. Global gene expression assays derived from in vivo models for bone adaptation have identified a number of candidate genes and revealed potential load regulated pathways. Nevertheless, there are significant limitations when interpreting these data. The harvesting and analysis of large populations of osteocytes reports gene expression averaged over tens of thousands of cells, each of which reside in different micro-environments characterized by different levels of mechanical strain and local osteoblastic/osteoclastic activity. It is therefore possible that key genes and networks are being concealed. Emerging studies investigate local regulation of gene expression in osteocytes by comparing 2D histology sections from loaded bone stained for specific molecular targets (sclerostin) with micro finite element (μFE) models. Whilst informative, these approaches are still very much qualitative and only permit the analysis of one specific molecular target at a time.

Addressing these limitations are microfluidic imaging approaches which allow for spatial and temporal mapping in three dimensions and quantitative measurement of gene expression cells in an organized “organ-on-a-chip” niche. Examples of a “microfluidic imaging” approach can be briefly described by the following workflow: bone formation and/or resorption are spatially mapped and quantified in technologies such as in vivo μCT and 3D image registration techniques; labeling (e.g., fluorescence, radio labeling) or other techniques, (e.g., chemical exchange saturation transfer (CEST), pH measurement T1rho, magnetization transfer contrast, magnetization exchange or other technologies. The vast amount of data generated using these approaches can be used to build, feed and validate computational models of various skeletal and other tissues, which incorporate all of the different length scales, from the organ-level to the cellular-level. Further examples include those described in Trussel et al., “Toward mechanical systems biology in bone.” Ann Biomed Eng. 2012 November; 40(11):2475-87.

Described herein is a method of detecting properties of one of more cells in a microfluidic device. In other embodiments, the microfluidic device includes mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes. In other embodiments, the microfluidic device includes cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. It is emphasized that the described methods and techniques find wide applicability to biological tissues. In other embodiments, the microfluidic device includes stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In various embodiments, the properties are biochemical properties of the one or more cells in a microfluidic device.

In various embodiments, the method includes providing a microfluidic device, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to one or more biochemical properties of one or more cells in the microfluidic device. In other embodiments, one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.

In various embodiments, the method includes applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more biochemical properties.

In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further culturing of cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells among others. In other embodiments, the method includes further detection of pulse sequence signal intensity. In various embodiments, the method includes detecting cellular mineralization. In other embodiments, the method includes detecting secreted extracellular macromolecules. In various embodiments, the method includes detecting cellular survival, differentiation and/or proliferation.

Described herein is a method of detecting cellular mineralization in a microfluidic device including providing a microfluidic device including mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes, adding one or more labeling agents to the microfluidic device, and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, the one or more labeling agents comprise bisphosphonate imaging agents. In other embodiments, the bisphosphonate imaging agent includes a pamidronate backbone with a fluorescent label. In other embodiments, the one or more labeling agents comprise a radiolabel. In other embodiments, the radiolabel includes technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD). In other embodiments, detecting the labeling agent includes Micro CT, Micro SPECT, and/or PET imaging. In other embodiments, detecting the labeling agent further includes comparison of the quantity of detected labeling agent with one or more control samples. In other embodiments, the method includes further culturing of MSCs, osteoblasts and/or osteocytes in the microfluidic device. In other embodiments, the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device. In other embodiments, the method includes further detection of the labeling agent.

Also described herein is method of detecting secreted extracellular macromolecules in a microfluidic device including providing a microfluidic device including stem cells, applying one or more pulse sequences to the microfluidic device, and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells. In other embodiments, the stem cells are mesenchymal stem cells (MSCs). In other embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In other embodiments, detecting the pulse sequence signal intensity includes chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX). In other embodiments, CEST detects a quantity of glycosaminoglycans (GAGs). In other embodiments, pH measurement of T1 rho detects a quantity of GAGs. In other embodiments, MTC detects a quantity of collagen. In other embodiments, MEX detects a quantity of collagen and/or osteoid. In other embodiments, the microfluidic device further includes one or more channels for loading of a control sample. In other embodiments, detecting the pulse sequence signal intensity further includes comparison of the quantity of detected pulse sequence signal intensity with one or more control samples. In other embodiments, the method includes further culturing of stem cells in the microfluidic device. In other embodiments, the method includes further culturing cartilage, tendon/ligament, nucleus pulposus, annulus fibrosus, chondrocytes, tenocytes, fibroblasts, and/or notochordal cells in the microfluidic device. In other embodiments, the method includes further detection of pulse sequence signal intensity.

Example 1 Micro Imaging for Non Invasive Monitoring of Stem Cell-Induced Mineralization

Mesenchymal stem cells (MSCs) can differentiate to various skeletal cells including osteoblasts. A common assay of MSC differentiation to osteogenic cells includes measurements of mineralization within the culture. Several methods can be used to monitor mineralization over time in chips

Example 2 Labeling Agents for Stem Cell-Induced Mineralization

Fluorescence imaging—bisphosphonate imaging probes such as OsteoSense™ (Perkin Elmer) can be added to the chip at different time points, washed and then the chip is imaged in an optical scanner. Hydroxyapatite (HA) is a mineral form of calcium apatite and is the major mineral product of osteoblasts. Therefore, HA levels are a good biomarker for osteoblast activity. In addition, abnormal accumulation of HA can be indicative of a disease state. OsteoSense™ imaging agents bind with high affinity to HA. Since hydroxyapatite (HA) is known to bind pyrophosphonates and phosphonates as well as synthetic bisphosphonates with high affinity, OsteoSense™ agents were designed as bisphosphonate imaging agents. These probes consist of a pamidronate backbone functionalized with near-infrared fluorophore off the amino terminus of the R2 side chain. Specifically, OsteoSense™ imaging agents can be used to image areas of microcalcifications, bone remodeling and enables imaging of bone growth and resorption. The bisphosphonate probe attaches to micro calcifications and the fluorescent readout provides quantification of mineralization.

Example 3 Other Labeling Agents for Stem Cell-Induced Mineralization

Bisphosphonates (BPs; also known as diphosphonates), such as methylene diphosphonate (MDP) and zoledronic acid, can be labeled with technetium-99m ([99mTc]-BPs) for use in bone scintigraphy as has been used to detect osteoporosis and other skeletal-related events (SREs). These chemicals bind hydroxyapatite, which allow for imaging of bisphosphonates as described above. [18F]-Fluoride is another nuclide that is commonly used for bone imaging, and positron emission tomography (PET) and is believed to be superior to [99mTc]-BPs for the diagnosis of SREs.

Micro SPECT/PET imaging-99mTc-Methyl diphosphonate (Tc-MDP) can be added to the chip at different time points, washed and then the chip is imaged using a micro SPECT scanner. Alternative probes are [′8F)-Fluoride or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) [68Ga]BPAMD that can be imaged using a micro PET scanner. These probes also attach to mineralization foci and the uptake readouts can provide quantitative data of mineralization.

Example 4 Micro Imaging for Non Invasive Monitoring of Stem Cell-Induced Mineralization

Micro CT—high-resolution micro CT scanners can detect mineral particles as small as 500 nm. A non-destructive scan of the chip can provide an accurate measurement of mineralization generated by the developing tissues.

Example 5 Micro Imaging for Non Invasive Monitoring of Extracellular Macromolecules Secreted by Stem Cells

Different types of stem cells including MSCs and induced pluripotent stem cells (iPSCs) have been shown to differentiate to joint tissue cells such as osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells cells. While differentiating, the cells secret characteristic extracellular molecules such as aggrecan, glycosaminoglycans (GAGs), collagens and more.

A way to monitor the secretion of these molecules in a chip will include the use of micro MRI using different pulse sequences, including but not limited to: chemical exchange saturation transfer (CEST)—GAGs measurement; pH measurement T1 rho—GAGs measurement, magnetization transfer contrast (MTC)—collagen measurement, magnetization exchange (MEX)—collagen and osteoid measurement.

Chemical exchange saturation transfer (CEST) also provides the ability to analyze the GAG content in cartilage. The most common method for acquisition of a CEST data set is to acquire multiple image data sets with presaturation at different offset frequencies around the water resonance and one reference data set without saturation or with saturation at a very large offset frequency. The normalized signal as a function of the presaturation offset (termed the z-spectrum) can then be used to determine and quantify CEST effects, which are asymmetric with respect to the water resonance (ie, a CEST effect appears either up- or down-field from water and therefore can be extracted from the z-spectrum via analysis of its asymmetry with respect to the water resonance).

Chemical exchange saturation transfer (CEST) is a magnetic resonance imaging (MRI) contrast enhancement technique that enables indirect detection of metabolites with exchangeable protons. Endogenous metabolites with exchangeable protons including many endogenous proteins with amide protons, glycosaminoglycans (GAG), glycogen, myo-inositol (MI), glutamate (Glu), creatine (Cr) and several others have been identified as potential in vivo endogenous CEST agents. These endogenous CEST agents can be exploited as non-invasive and non-ionizing biomarkers of disease diagnosis and treatment monitoring.

Magnetization Transfer Contrast (MTC) MRI is an imaging method that evolved from NMR spectroscopy. In tissue imaging, MTC relies upon the interaction of less mobile protons associated with macromolecules such as proteins and their interactions with protons freely associated with water. The premise is that in a system where molecules move and exchange position, whether it be a change in spatial position in asymmetrical molecules or an exchangeable proton between a molecule and water, the magnetization state will also move and be transferred.

A two pool model can be utilized to illustrate the theory behind MTC MM. Conventional MRI detects only the free water pool while the macromolecular pool remains mostly undetected. Both the macromolecular and free water pools are centered around the same frequency but the macromolecular pool is shallower and wider. Saturation is achieved by applying an off-resonance radio frequency (RF) pulse specific to a peak in the macromolecular pool before excitation at the center frequency. The RF pulse saturates the signal from the section leading to ideally no signal at the off-resonance frequency. Since both pools interact this saturation is transferred to the free water pool. While it is not possible to detect the changes in the macromolecular pool directly, it can be assumed that the loss in signal intensity of the free water pool corresponds to the changes in the macromolecular pool.

Ideally, an increase is preferable to a decrease in signal intensity since it is easier to visualize changes in brightness over changes in darkness. To achieve this type of image, a Magnetization Transfer Ratio (MTR) is calculated using a base image without saturation to measure the relative loss of signal intensity in a pixel by pixel basis: MTR=Nonsaturated−Saturated/Nonsaturated. MTC is very similar in function to CEST. CEST focuses on a limited part of magnetization transfer by linking it to chemical exchange systems.

Quantitative magnetization transfer (qMT) imaging is MR technique which utilizes a two-pool model of magnetization exchange to acquire information regarding the cartilage macromolecular matrix. qMT imaging techniques typically require multiple MT-contrast images with different magnetization preparatory pulses resulting in long scan times which have limited cartilage assessment to ex-vivo specimens. Cross-relaxation imaging (CRI) is a qMT method which can create three-dimensional parametric maps of articular cartilage measuring the fraction of macromolecular bound protons (f), the exchange rate constant between macromolecular bound protons and free water protons (k), and the T2 relaxation time of macromolecular bound protons (T2B) with high resolution and relatively short scan time based upon a limited number of MT-contrast images. The parameter f provides an indirect measure of macromolecular content, while the parameters k, and T2B reflect the efficiency of magnetization exchange between macromolecular bound protons and free water protons and the spin diffusion between proton sites in macromolecules respectively which may be influenced by macromolecular organization and ultra-structure

Example 6 Micro Imaging for Non Invasive Monitoring of Osteogenic Cells-On-Chip

Microfluidic culture devices are attractive systems to model physiological and pathological conditions of tissues and organs. Although these devices allow fluorescent and light microcopy imaging of cultured cells, one of its current limitations is that various types of analyses require sacrificing of the culture. The Inventors have previously utilized micro imaging systems to monitor stem cell differentiation in ex-vivo 3D tissue constructs.

Of interest is utilizing optical imaging to non-invasively monitor stem cell survival and differentiation while cultured in an “organ-on-chip” device. Stiffer membrane and microfluidic environment will promote more efficient osteogenic differentiation.

To explore this possibility, the organ-on-chip was coated with ECM crosslinked with UV prior to cell seeding. Then mesenchymal stem cell line overexpressing BMP2 and Luciferase reporter genes were seeded on the coated organ-on-a-chip (see dimensions and the set up for microfluidic studies in FIG. 1 and supplemented with osteogenic media. The static cultures were performed using 200 μl media reservoirs that were changed every other day. The flow studies were performed using 30 μl/h flow of media pulled through using specialized pump (FIG. 1). Micrographs were taken twice a week and survival of the cells was monitored using bioluminescent imaging. The media was changed to media with Luciferin and imagined using IVIS (Perkin Elmer). The osteogenic differentiation after 3 weeks of culture in osteogenic media was monitored using florescent probes OsteoSense650 and BoneTag800 that were introduced 24 hours before the imaging and were imaged using fluorescent imaging (FLI) and near infrared (NIR) imaging, confocal microscopy and immunostaining

Example 7 Monitoring Cell Proliferation without Harvest or Culture Disruption

A comparison of chips grown in static culture condition to chips grown under constant flow of media (30 μl/h) was performed along with evaluation of the effect of the flow on cell survival/proliferation of cells and the extent of osteogenic differentiation. The microscopic images (FIG. 1A) show proliferation of the cells under the flow conditions, however it is difficult to quantify the extent of proliferation using this method without disrupting the cultures. Therefore, the Inventors used cell that express Luciferase reporter gene and the cell proliferation was quantified using bioluminescent imaging (BLI) twice a week (FIG. 2B, C). This imaging method allowed monitor the proliferation of the cells without the need to harvest or disrupt the culture and significant advantage to the flow system was observed. Also microfluidic environment had positive effect on osteogenic differentiation, when compared with static cultures.

This effect was observed in fluorescent imaging of osteogenic differentiation probes using two different systems—FLI and Near Infrared (FIG. 3). The probes can be detected using different wavelengths of fluorescence, therefore both probes can be added simultaneously and imaged separately. The quantification of FLI (FIG. 3A, B) of BoneTag showed higher osteogenic differentiation of the cell under the flow conditions. OsteoSense was also imaged using confocal microscopy in conjugation with Live/Dead staining (FIG. 3C) showing that most of the live cells absorbed OsteoSense probe and again the flow chips were stained in more efficiently than the static cultures. The NIR system is considered more sensitive and the quantification of the image more accurate. Here, the Inventors demonstrate that they system is capable of detecting the same trend using both probes (FIG. 3D).

Example 8 Confirmation of Osteogenic Differentiation

In order to confirm osteogenic differentiation of the MSSC-BMP2 cells, the harvested chips were sectioned using vibratome creating transvers sections across the channels. Then these sections were subjected to immunofluorescent staining using primary antibody against Osteocalcin and Bone Sialoprotein (BSP) osteogenic markers. The staining shows cells on both sides of the membrane in both conditions, but mainly in the top channel. In both conditions there was positive staining for both marker, indicating osteogenic differentiation, however the staining looks more prominent in the chips that were cultured in flow (FIG. 4 bottom panel).

Organ-on-chip system allows monitoring of the cell survival and proliferation in vitro using BLI imaging system and monitor the osteogenic differentiation of the cell on the chip in real time, without the need of harvesting the cells and disrupting the culture conditions. Here, the Inventors demonstrate that the flow conditions affect both proliferation and the differentiation of the MSCs that overexpress BMP2.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are sources of mesenchymal stem cells, osteoblasts, bone cells or stem cells, seeding and culturing on a microfluidic device, imaging methods, including labeling and detection, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described. 

1. A method of detecting cellular mineralization in a microfluidic device comprising: providing a microfluidic device comprising mesenchymal stem cells (MSCs), osteoblasts and/or osteocytes; adding one or more labeling agents to the microfluidic device; and detecting the labeling agent, wherein the labeling agent is capable of binding to cellular mineralization.
 2. The method of claim 1, wherein the microfluidic device further comprises one or more channels for loading of a control sample.
 3. The method of claim 1, wherein the one or more labeling agents comprise bisphosphonate imaging agents.
 4. The method of claim 3, wherein the bisphosphonate imaging agent comprises a pamidronate backbone with a fluorescent label.
 5. The method of claim 3, wherein the one or more labeling agents comprise a radiolabel.
 6. The method of claim 5, wherein the radiolabel comprises technetium-99m ([99mTc]-BPs), [18F]-Fluoride, 99mTc-Methyl diphosphonate (Tc-MDP), and/or 68Ga-Labeled (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,1 O-bis(carboxymethyl)-I,4, 7, I 0-tetraazacyclododec-1-yl)acetic acid (BPAMD) ([68Ga]BPAMD).
 7. The method of claim 1, wherein detecting the labeling agent comprises Micro CT, Micro SPECT, and/or PET imaging.
 8. The method of claim 1, wherein detecting the labeling agent further comprises comparison of the quantity of detected labeling agent with one or more control samples.
 9. The method of claim 1, comprising further culturing of mesenchymal stem cells (MSCs), osteoblasts, osteocytes, chondrocytes, tenocytes, fibroblasts, notochordal cells, and/or nucleus pulposus cells in the microfluidic device.
 10. The method of claim 9, comprising further detection of the labeling agent.
 11. A method of detecting secreted extracellular macromolecules in a microfluidic device comprising: providing a microfluidic device comprising stem cells; applying one or more pulse sequences to the microfluidic device; and detecting the pulse sequence signal intensity, wherein the pulse sequence signal intensity is capable of measuring one or more macromolecules secreted by the stem cells.
 12. The method of claim 11, wherein the stem cells are mesenchymal stem cells (MSCs).
 13. The method of claim 11, wherein the stem cells are pluripotent stem cells (pSCs).
 14. The method of claim 13, wherein the stem cells are induced pluripotent stem cells (iPSCs).
 15. The method of claim 11, wherein detecting the pulse sequence signal intensity comprises chemical exchange saturation transfer (CEST), pH measurement of T1 rho, magnetization transfer contrast (MTC), and/or magnetization exchange (MEX).
 16. The method of claim 15, wherein CEST detects a quantity of glycosaminoglycans (GAGs).
 17. The method of claim 15, wherein pH measurement of T1 rho detects a quantity of GAGs.
 18. The method of claim 15, wherein MTC detects a quantity of collagen.
 19. The method of claim 15, wherein MEX detects a quantity of collagen and/or osteoid.
 20. The method of claim 11, wherein the microfluidic device further comprises one or more channels for loading of a control sample.
 21. The method of claim 11, wherein detecting the pulse sequence signal intensity further comprises comparison of the quantity of detected pulse sequence signal intensity with one or more control samples.
 22. The method of claim 11, comprising further culturing of stem cells in the microfluidic device.
 23. The method of claim 21, comprising further detection of pulse sequence signal intensity. 